The use of bacterial cells to produce recombinant peptides is increasing in commercial importance. One of the goals in developing a bacterial expression system is the production of high quality target polypeptides quickly, efficiently, and abundantly. An ideal host cell for such an expression system would be able to efficiently utilize a carbon source for growth, efficiently produce a target polypeptide, quickly grow to high cell densities in a fermentation reaction, express the target polypeptide only when induced, and allow for the induction of the target polypeptide in an inexpensive and efficient manner.
One hurdle to the creation of the ideal host cell is overcoming inefficient and low level production of target polypeptides in the fermentation process. Controlling expression of the target peptide until optimal host cell densities and fermentation conditions are reached allows for a more efficient and larger yield of polypeptide. The reasons for this are several fold, including a more efficient utilization of a carbon source and the reduction of extended metabolic stresses on the host cell.
One way to control expression of target polypeptides during the fermentation process includes the use of inducible or regulatable promoters to control expression of the peptides. Promoters are regulatory nucleic acid sequences generally located upstream from the desired peptide coding sequence, directing the transcription of the nucleic acid. Promoters are generally classified as constitutive or regulated promoters. Regulated promoters include: (1) activatable promoters, which are inactive until an activator peptide binds to the 5′ regulatory regions; and (2) repressible promoters, which are inactive while the 5′ regulatory region is bound by a repressor peptide. Some genes or operons are regulated by more than one mechanism.
An inducible or regulatable promoter can be an essential component for the production of high levels of recombinant peptides in an efficient manner, since such regulatable promoters allow for cell densities to reach optimal levels prior to the induction of peptide production. Attributes of an ideal promoter can include: tight repression so that little or no target peptide is made during the growth phase; strong promoter activity after the addition of the inducer; low cost of induction; stability of the inducer in the cell culture medium so that the inducer need only be added once during the peptide accumulation phase of a fermentation process; easy passage of the inducer of the promoter through the cell membrane so that the external concentrations of the inducer are directly related to the internal concentration within the cell, and linearly related to peptide production; and ability to be used in tandem with other promoters. Such an ideal promoter allows recombinant peptides to be induced at high levels efficiently and inexpensively.
One regulatable promoter that has found widespread use in bacterial fermentation process for the production of recombinant peptides is the lac promoter, and its derivatives, especially the tac and trc promoters. In commercial fermentation systems using a lac-type promoter, the inducer isopropyl-β-D-1-thiogalactopyranoside (“IPTG”) is almost universally employed. IPTG is, however, expensive and must be carefully controlled since it is significantly toxic to biological systems. Standard IPTG preparations are currently available at about USD $18 per gram or about USD $125 per 10 grams. In addition, standard IPTG preparations may contain dioxane, a toxin. Dioxane-free IPTG is available on the market, but costs roughly twice the price of standard IPTG. Furthermore, environmental and health regulatory issues arise in regard to the presence of IPTG in the fermentation, or in the protein purified from the fermentation, given the IPTG toxicity risks to humans, animals, and other biological organisms.
Because of the toxicities and costs associated with IPTG, alternative inducible promoter systems have been proposed for use in bacterial fermentation processes for the production of recombinant peptides. For example, promoters induced by high temperatures such as λPR and λPL, tryptophan starvation such as trp, 1-arabinose such as araBAD, phosphate starvation such as phoA, nalidixic acid such as recA, osmolarity such as proU, glucose starvation such as cst-1, tetracycline such as tetA, pH such as cadA, anaerobic conditions such as nar, T4 infection such as T4 gene32, alkyl- or halo-benzoates such as Pm, alkyl- or halo-toluenes such as Pu, salicylates such as Psal, and oxygen such as VHb, have all been examined as alternatives to IPTG inducible promoters. See, for example, Makrides, S. C. (1996) Microbiol. Rev. 60, 512-538; Hannig G. & Makrides, S. C. (1998) TIBTECH 16, 54-60; Stevens, R. C. (2000) Structures 8, R177-R185; J. Sanchez-Romero & V. De Lorenzo, Genetic Engineering of Nonpathogenic Pseudomonas strains as Biocatalysts for Industrial and Environmental Processes, in Manual of Industrial Microbiology and Biotechnology (A. Demain & J. Davies, eds.) pp. 460-74 (1999) (ASM Press, Washington, D.C.); H. Schweizer, Vectors to express foreign genes and techniques to monitor gene expression for Pseudomonads, Current Opinion in Biotechnology, 12:439-445 (2001); and R. Slater & R. Williams, The Expression of Foreign DNA in Bacteria, in Molecular Biology and Biotechnology (J. Walker & R. Rapley, eds.) pp. 125-54 (2000) (The Royal Society of Chemistry, Cambridge, UK). Several problems exist with these types of promoters. For example: high temperature induction may be harmful to cells, and may not be practical for large scale fermentation due to equipment limitations; oxygen manipulation may affect the overall dynamics of the cell growth density aspects of the fermentation, reducing ideal conditions; the use of toluenes or other similar types of potentially toxic chemicals may require further purification to ensure that these compounds are not present in the final product; and pH may affect the ability of the peptide of interest to correctly fold or be solubilized in the host, making purification more costly and difficult.
Promoters that can be induced by inexpensive and non-toxic carbon sources remain attractive for use in bacterial fermentation processes. One potential advantage to such a promoter is that bacterial host may have endogenous mechanisms to efficiently uptake the inducer. Potential carbon sources that can be used as inducers include maltose, maltodextrin, glucose, arabinose, fructose, galactose, sucrose, glycerol, mannose, acetate, and lactose. Other potential carbon source inducers include the alcohol forms of carbon sugars, such as mannitol, glucitol, and arabitol.
Mannitol as Potential Inducer
Mannitol is the alcohol form of mannose, and is an inexpensive carbon source for a number of bacteria, including Pseudomonads. The operon involved with uptake and degradation of mannitol in Pseudomonas fluorescens (P. fluorescens) contains seven genes, four of which (mtlEFGK) encode proteins involved in mannitol/glucitol/arabitol uptake and transport, and three of which (mtlDYZ) encode proteins involved in the catabolism of mannitol, glucitol, and arabitol. See Brunker et al. (1998) “Structure and function of the genes involved in mannitol, arabitol, and glucitol utilization from Pseudomonas fluorescens DSM50106,” Gene 206(1):117-126 and FIG. 1.
Brunker et al. have further identified a sequence similar to the consensus for E. coli sigma 70 promoters 90 bp upstream from the start codon of the first gene in the P. fluorescens DSM50106 operon, mtlE. See FIG. 2. A 660 bp fragment containing the putative promoter was cloned upstream of a luciferase gene upon which it conferred mannitol-inducible expression in E. coli. Arabitol induced expression of the gene to the same level, and glucitol induced expression to a level half as high.
The benefits of carbon source inducible promoters, however, is not without its limitations. One potential problem associated with utilizing carbon source inducible promoters is the ability of the host cell to metabolize the inducer and reduce the effectiveness of the inducer, or require it to be continually added to the media during induction. In addition, the inducer may compromise carbon utilization parameters of the fermentation process, and the constant inducer flux may result in less than desirable peptide production yields. The requirement of continually adding an inducer to the media during induction has the further disadvantage of increasing the cost of the fermentation process.